ANIMAL BEHAVIOUR, 2002, 64, 697–708 doi:10.1006/anbe.2002.4011, available online at http://www.idealibrary.com on

Spatiotemporal patterns of intraspecific aggression in the invasive Argentine

ANDREW V. SUAREZ*‡, DAVID A. HOLWAY*, DANGSHENG LIANG†, NEIL D. TSUTSUI*‡ & TED J. CASE* *Division of Biological Sciences, Section of , Behavior & Evolution, University of California San Diego †Clorox Technical Center, Pleasanton, California, U.S.A. ‡Center for Population Biology, Section of Ecology & Evolution, University of California Davis

(Received 29 August 2001; initial acceptance 9 November 2001; final acceptance 3 April 2002; MS. number: A9148)

The Argentine ant, Linepithema humile, is a widespread invasive characterized by reduced intraspecific aggression within introduced populations. To illuminate the mechanisms underlying nestmate recognition in Argentine , we studied the spatial and temporal fidelity of intraspecific aggression in an introduced population of Argentine ants within which intraspecific aggression does occur. We quantified variation in the presence or absence of intraspecific aggression among nests over time both in the field and under controlled laboratory conditions to gain insight into the role of environmental factors as determinants of nestmate discriminatory ability. In addition, we compared levels of intraspecific aggression between nest pairs to the similarity of their cuticular hydrocarbons to determine the potential role of these compounds as labels for nestmate discrimination. In both field and laboratory comparisons, nest pairs behaved in a consistent manner throughout the course of the experiment: pairs that fought did so for an entire year, and pairs that did not fight remained nonaggressive. Moreover, we found a negative relationship between cuticular hydrocarbon similarity and the degree of aggression between nests, suggesting that these hydrocarbons play a role in nestmate discriminatory ability. In contrast to the prevailing pattern, ants from one site showed a marked change in behaviour during the course of this study. A concomitant change was also seen in the cuticular hydrocarbon profiles of ants from this site.  2002 The Association for the Study of Behaviour. Published by Elsevier Science Ltd. All rights reserved.

In social , systems for nestmate recognition are in ants (Breed & Bennett 1987; Ho¨lldobler & often well developed, allowing colony boundaries to be Wilson 1990; Banschbach & Herbers 1996; Beye maintained with high fidelity. Because social et al. 1998; Stuart & Herbers 2000). colonies are typically groups of related individuals, non- Many insects use cuticular hydrocarbons to recognize reproductives gain inclusive fitness by preferentially help- mates, conspecifics and colony members (Howard & ing nestmates (Hamilton 1964). Cues used to distinguish Blomquist 1982; Vander Meer & Morel 1998). As a result, nestmates from non-nestmates may be environmentally variation in cuticular hydrocarbons has been used to derived, innate (genetically based), or a combination of identify species (Howard et al. 1982; Vander Meer & both. Using these cues, individuals can discriminate nest- Lofgren 1990), to differentiate populations (Nowbahari mates from non-nestmates, and thus accept or reject et al. 1990), and to distinguish among different individuals they encounter. Environmental odours, such castes within social insect colonies (Howard et al. 1982; as those acquired from food or nesting material, can be Haverty et al. 1996). Intercolonial aggression in ants used to assess group membership in many , , also correlates with variation in cuticular hydrocarbon , and some ants (Ho¨lldobler & Michener 1980; profiles (Bonavita-Cougourdan et al. 1987; Nowbahari Gamboa et al. 1986; Carlin 1989; Ho¨lldobler & Wilson et al. 1990). Moreover, recent experiments suggest 1990; Breed 1998). Current evidence suggests that that cuticular hydrocarbons directly affect nestmate genetically based systems are also important, particularly recognition in some ant species (Lahav et al. 1999; Thomas et al. 1999; Boulay et al. 2000; Liang & Silverman Correspondence and present address: A. V. Suarez, University of California, Department of Environmental Science, Policy and 2000). Management, Division of Insect Biology, 201 Wellman Hall #3112, Because ants show a variety of colony structures, they Berkeley, CA 94720-3112, U.S.A. (email: [email protected]. have long been used as model organisms to examine the edu). mechanisms underlying recognition systems (Carlin & 697 0003–3472/02/$35.00/0  2002 The Association for the Study of Animal Behaviour. Published by Elsevier Science Ltd. All rights reserved. 698 ANIMAL BEHAVIOUR, 64, 5

Ho¨lldobler 1986; Ho¨lldobler & Wilson 1990). In many In this paper, we examined the temporal fidelity of ants, nestmate discrimination is well developed and nestmate recognition among nests of Argentine ants that workers aggressively defend territories, particularly vary in the degree to which they show intraspecific against conspecifics, resulting in a of colony struc- aggression. We studied changes in behaviour among ture known as multicoloniality (Ho¨lldobler & Wilson nests over time in the field and under controlled 1977). In contrast, some species show a colony structure laboratory conditions in order to gain insight into the known as unicoloniality in which levels of intraspecific role of environmental factors as determinants of nest- aggression are reduced or absent and colony boundaries mate discriminatory ability. In addition, we compared are weak to nonexistent (Ho¨lldobler & Wilson 1977). levels of intraspecific aggression between nest pairs to the Unicoloniality is rare in ants, and is most commonly similarity of their cuticular hydrocarbons to investigate observed in mound-building wood ants (of the their potential role in nestmate discrimination. rufa group) and many invasive ants, including the Argentine ant, Linepithema humile (Ho¨lldobler & Wilson 1990; Passera 1994; Bourke & Franks 1995). METHODS Native to South America, Argentine ants have become established in Mediterranean and subtropical climates Selection of Field Sites throughout the world (Suarez et al. 2001; Tsutsui et al. 2001) where they commonly displace native ants (Bond To investigate temporal variation in nestmate discrimi- & Slingsby 1984; Ward 1987; Cammell et al 1996; natory ability in the Argentine ant, we identified nine Human & Gordon 1996; Holway 1998; Suarez et al. invaded sites in southern California that varied in the 1998). Within native populations, Argentine ants typi- degree to which Argentine ants showed intraspecific cally appear more multicolonial, with intraspecific aggression. Because intraspecific aggression is rare in the aggression frequently occurring over short (<100 m) introduced range, extensive surveys throughout southern spatial scales (Suarez et al. 1999; Tsutsui et al. 2000). California were conducted between 1997 and 1999 to Within introduced populations, however, intraspecific identify colonies among which intraspecific aggression aggression is almost entirely absent and Argentine ants occurred (Fig. 1; Suarez et al. 1999; Tsutsui et al. 2000). form expansive supercolonies (Newell & Barber 1913; Colonies from most of these locations have been used Markin 1968; Ho¨lldobler & Wilson 1990; Wayetal. previously to investigate the genetic correlates (Tsutsui 1997; Suarez et al. 1999; Tsutsui et al. 2000; Giraud et al. 2000) and colony-level consequences (Holway et al. et al. 2002). It has been suggested that unicolonialty has 1998) of intraspecific aggression. In August 1999, we arisen in introduced populations of the Argentine ant as collected workers from one nest at each of nine sites and a result of reduced genetic diversity following their performed pairwise behavioural assays between workers introduction (Tsutsui et al. 2000). Introduced popula- from all the sites (N=36 nest pairs). In addition, we tions may not possess the levels of genetic diversity collected workers, brood and queens from eight sites to necessary to distinguish nestmates from non-nestmates. establish laboratory colonies. Each month for 12 months, This variation in colony structure may also underlie the we returned to each site of collection and repeated Argentine’s ability to displace native species. Specifi- behavioural assays among all nest pairs. cally, the numerical advantages key to the Argentine ant’s success in displacing native species (Holway 1999; Holway & Case 2001) may result from the reduced Maintenance of Laboratory Colonies mortality, higher foraging rates and greater brood pro- duction resulting from an abandonment of costly We established laboratory colonies of Argentine ants intraspecific territorial defence (Holway et al. 1998). from the eight field nests in August 1999, following the Given the lack of intraspecific aggression typical of protocol of Holway et al. (1998). Each colony occupied a introduced populations of Argentine ants, and its plastic nest container (30148 cm) lined with Fluon potential role in their success, it is of great interest to (Northern Products Inc., Woonsocket, Rhode Island, determine the mechanisms responsible for nestmate U.S.A.) and Tanglefoot (The Tanglefoot Company, recognition in this species. Recent work on Argentine Grand Rapids, Michigan, U.S.A.) to prevent ants from ants found a negative relationship between the extent escaping. Each nest container held three test-tubes half- of intraspecific aggression and the degree of genetic filled with water and plugged with cotton to serve as nest similarity between nests in both native and introduced chambers. We started colonies with at least 1000 workers, populations (Tsutsui et al. 2000). This finding suggests 100 brood pieces, and a queen to worker ratio similar to that nestmate recognition cues are heritable. However, that in the field at the time of collection (1–10 queens/ environmental factors also affect nestmate recognition in laboratory colony). Colonies were fed crickets or scram- Argentine ants. For example, acquisition of cuticular bled eggs every 3 days and sugar water daily. We hydrocarbons from one prey species, the brown-banded performed behavioural assays to measure aggression cockroach, Supella longipalpa, induces intraspecific aggres- among all pairwise combinations of the eight nests sion between nestmates in a laboratory setting (Liang & (N=28) each month for 11 months starting 1 month after Silverman 2000). Such prey-induced aggression can collection. We also performed behavioural assays each result in colony disassociation after long-term exposure month between the laboratory colonies and the field nest (Silverman & Liang 2001). from which they were collected. SUAREZ ET AL.: AGGRESSION AMONG ARGENTINE ANT NESTS 699

Lake Skinner A 15 km Lake Skinner B 150 km Temecula

Lake Hodges A 33.2N Lake Hodges B California, U.S.A.

32.8N Coast Mesa Sweetwater 3B

Sweetwater 3A San Diego *

Mexico

117.4W 117.0W Figure 1. Map of California with an inset of southwestern California showing the locations of Argentine ant populations screened for intraspecific aggression. Intraspecific aggression was never detected among sites with the same symbol nor within sites regardless of symbol. The nine field locations used to examine temporal variation in intraspecific aggression in Argentine ants are labelled.

Behavioural Assays equipped with a flame-ionization detector and interfaced with a HP GC ChemStation data acquisition system We quantified intraspecific aggression between nests (Rev.A.05.02). Following splitless injection of the sample, using a simple behavioural assay (Carlin & Ho¨lldobler we maintained the temperature at 80 C for 2 min, 1986; Holway et al. 1998). For each trial, two workers, one increased the temperature to 270 Cat20C/min, and from each of two nests, were placed into a 2-dram    then increased the temperature to 310 Cat3C/min. (3.7 ml) plastic vial coated with Fluon . We scored Both detector and injector were maintained at 320 C. behavioural interactions between the two workers for 5 min using the following categories in order of escalating aggression: touch=1 (contacts that included prolonged Statistical Analyses antennation); avoid=2 (contacts that resulted in one or Nest pairs fell into two groups at the onset of the both ants retreating rapidly in opposite directions); experiment: pairs that showed aggression and pairs that aggression=3 (a brief, <2 s, expression of any of the did not. While fighting trials were occasionally scored as following behaviours: lunging, biting and pulling legs a 2 (avoiding behaviour) or a 3 (aggressive behaviours or antennae, use of chemical defensive compounds); present but not sustained), the majority of the trials were or fight=4 (prolonged aggression between individuals scored as eithera1or4(sustained fighting). Subse- usually resulting in death or dismemberment). At the end quently, we considered a nest pair to be aggressive if at of 5 min, we recorded the maximum score attained dur- least one of the five trials escalated to sustained fighting. ing the trial; most trials were scored as eithera1ora4. We used a repeated measures multivariate analysis of The mean value for each set of five trials was used in variance (MANOVA, using JMP, SAS Institute 1996)to subsequent analyses. compare aggression levels between field and laboratory nest pairs over the 11 months of the study. We conducted Extraction and Isolation of Cuticular Hydrocarbons four separate MANOVAs: (1) laboratory versus field (aggressive pairs), (2) laboratory versus field (nonaggres- Each month, starting in August 1999, we collected sive pairs), (3) aggressive versus nonaggressive pairs (field 25–100 workers from each field nest and immediately nests), and (4) aggressive versus nonaggressive (laboratory placed them on ice. In the laboratory, we maintained nests). For each MANOVA, dependent variables were samples at 20 C until analysis. To extract cuticular aggression scores for each experimental group at each hydrocarbons, we washed 5–10 ants from each sample in time period. hexane for 10 min followed by a brief secondary rinse. Numerous studies have reported decreases in intra- The solution was loaded onto a silica gel (60–200 mesh, specific aggression over time when colonies are reared in J. T. Baker, Philipsberg, New Jersey, U.S.A.) minicolumn, the laboratory (Stuart 1987; Breed 1998; Boulay et al. and hydrocarbons were then eluted with 5 ml of hexane. 2000; Chen & Nonacs 2000). To determine whether this We analysed hydrocarbon profiles using 30 m=0.32 mm occurred in our laboratory colonies, we used regression to DB-5 capillary columns with an HP589011 or HP5880 GC assess whether levels of aggression decreased through 700 ANIMAL BEHAVIOUR, 64, 5

time for each nest (corrected for multiple comparisons, 4 Rice 1989). To avoid problems of independence that may (a) arise by including each nest pairing in the analysis, we averaged the mean aggression scores across all aggressive pairings each month to use as the dependent 3 variables. To examine the relationship between cuticular hydro- carbon similarity and intraspecific aggression, we con- 2 structed a matrix with each cell containing the pairwise correlation coefficient of hydrocarbon profiles between nests. We used the relative contribution (i.e. height) of the largest 42 peaks as a quantitative measure of each 1 profile. For each nest, we replicated the extraction three to four times, and the mean value for each of the largest 42 peaks was used in subsequent analyses. Each corre- 4 lation coefficient used in the matrix compared the (b) heights of each of 42 peaks for each nest pair. Second, we constructed a matrix of pairwise aggression scores and used a Mantel test (using Arlequin 2.000; Schneider 3 et al. 2000) with 1 million permutations of the data) to

compare the two matrices. Finally, for two field sites aggression between nest pairs Average (Mesa and Sweetwater 3A) we examined temporal vari- ation in cuticular hydrocarbons by comparing correlation 2 coefficients (as described above) across four time periods from August 1999 to August 2000. These nests were chosen because Sweetwater 3A demonstrated a change in 1 behaviour throughout the course of the study (see Results below). For each comparison, we used individual replicates (rather than means) to determine a mean July May June (1 SD) correlation coefficient for each time period. April March August August October January

Previous work in this system has documented a relation- February December November ship between variation in cuticular hydrocarbons and September aggression (Liang & Silverman 2000; Silverman & Liang Figure 2. Summary of behavioural results for field and laboratory 2001); hydrocarbons with a response time between 20 nest pairs. (a) All data for field (open symbols)(not including and 30 min appear most important in determining Sweetwater 3A, see Results) and laboratory (filled symbols) nest pairs categorized into two groups based on the presence (squares) or behavioural variation. absence (circles) of aggression between nest pairs at the onset of the study. (b) The same data as in (a) but also including field nest pairs RESULTS involving Sweetwater 3A demonstrating the change of behaviour of this nest after January. Error bars represent one standard error. Behavioural Patterns Both in field and laboratory comparisons, the behav- iour of nest pairs remained consistent throughout the removed from the overall analysis (MANOVA: field

course of the experiment: aggressive pairs remained versus laboratory Wilk’s lamda=0.6202, F1,10=0.0612, aggressive, nonaggressive pairs remained nonaggressive P=0.9917) (Fig. 2a). Moreover, laboratory nest pairs (Fig. 2a). The only exception to this general pattern was that did not show intraspecific aggression at the start of the Sweetwater 3A field site. While workers from this site the experiment never consistently developed aggression were initially nonaggressive towards ants from a subset of (Figs 2, 4). sites (Mesa, Coast and Sweetwater 3B), after January 2000, As with nonaggressive pairs, the overall behavioural workers collected from Sweetwater 3A were, in almost all pattern between aggressive pairs was consistent, despite cases, aggressive towards workers from all sites (Fig. 2b, some temporal variation. Aggressive nest pairs main- Fig. 3). tained higher assay scores than nonaggressive pairs MANOVA results confirmed the qualitative patterns in both for field (MANOVA: Wilk’s lamda=0.3672,

the data. For nests pairs that did not show aggression at F1,20=34.4682, P<0.0001) and laboratory compari- the onset of the experiment, there was no significant sons (MANOVA: Wilk’s lamda=0.1997, F1,19=76.1558, difference among field or laboratory nest pairs over time P<0.0001) (Fig. 2). Notably, levels of aggression among (MANOVA: field versus laboratory Wilk’s lamda=0.8259, laboratory colonies were slightly higher that those in the

F1,13=2.7407, P=0.1217; time: Wilk’s lamda=0.3567, field (MANOVA: Wilk’s lamda=0.8295, F1,26=5.3431,  F10,4=0.7212, P=0.6934; time location: Wilk’s lamda= P=0.029). Among aggressive pairs, levels of aggression 0.4845, F10,4=0.4255, P=0.8755) (Fig. 2b). This similarity varied with time (MANOVA: Wilk’s lamda=0.1990, was particularly evident when Sweetwater 3A was F10,17=6.8425, P=0.0003), and there was a significant SUAREZ ET AL.: AGGRESSION AMONG ARGENTINE ANT NESTS 701

4 Temecula Field comparisons 3 Behaviour between nest pairs at start of experiment 2 Aggression absent Aggression present 1

4 Coast Mesa

3

2

1

4 Sweetwater 3A Sweetwater 3B

3

2

1

4 Lake Skinner A Lake Skinner B Average aggression between nest pairs Average

3

2

1

4 Lake Hodges A Lake Hodges B

3

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1 July July May May June June April April March March August August August August October October January January February February December December November November September September Figure 3. Temporal patterns in intraspecific aggression of ants from each field colony when paired with all other sites. Behavioural comparisons between Sweetwater 3A and Mesa, Coast and Sweetwater 3B were excluded after January 2000 (see Results). Missing symbols indicate behavioural assays not conducted for that site during that month. Error bars represent one standard error. time by location (field or laboratory) interaction For a majority of nests in the laboratory, aggression,

(MANOVA: Wilk’s lamda=0.3442, F10,17=3.2390, when present, did not decline after 11 months (Fig. 4). P=0.0161). However, aggression did decline through time for three 702 ANIMAL BEHAVIOUR, 64, 5

Laboratory comparisons Behaviour between nest pairs at start of experiment Aggression absent Aggression present 4 Coast Mesa

3

2

1

4 Temecula Lake Hodges B

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4 Sweetwater 3A Sweetwater 3B

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2 Average aggression between nest pairs Average 1

4 Lake Skinner A Lake Skinner B

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1 July July May May June June April April March March August August January January October October February February December December November November September September Figure 4. Temporal patterns in intraspecific aggression of ants from each laboratory colony when paired with all other sites. Nests from three sites (Temecula, Skinner A and Sweetwater 3B) showed lower levels of aggression at the end of the experiment than at the start. Missing symbols indicate behavioural assays not conducted for that site during that month. Error bars represent one standard error.

nests (Fig. 4): Temecula (Y=3.7040.108X, R2=0.611, laboratory nests of Sweetwater 3A displayed aggression P=0.005), Skinner A (Y=3.4290.165X, R2=0.787, towards one another (Fig. 5). Aggression was rarely P<0.001), and Sweetwater 3B (Y=4.0040.145X, detected in the other comparisons until 10 months into R2=0.634, P=0.002). the experiment (Fig. 5). After this time, a few pairings (two of eight nests in June 2000, three of eight nests in Field versus laboratory comparisons July 2000) showed low mean levels of aggression. Across Behavioural assays between laboratory nests and the all nests, however, levels of aggression were not higher at field sites from which they were collected revealed no the end of the experiment than the beginning (paired  aggression until January 2000. At this time, the field and t test: t7= 1.879, P=0.1023). SUAREZ ET AL.: AGGRESSION AMONG ARGENTINE ANT NESTS 703

4 The change in behaviour seen in ants from the Sweetwater 3A field site was associated with a change in the hydrocarbon profiles of those ants. Correlation coefficients between the hydrocarbon profiles from ants 3 collected at Sweetwater 3A in August 1999 and from ants collected at later time points decreased after the change in behaviour. This change was not seen in ants from the Mesa site over the same time period (Fig. 8). Sample 2 profiles from Mesa and Sweetwater 3A also illustrate these differences (Fig. 9).

1 DISCUSSION

Average aggression between nest pairs Average When initially present, intraspecific aggression between nests of Argentine ants remained high over the course of 1 year, both in the field and under controlled conditions July 2000 May 2000 June 2000 April 2000 in the laboratory. Likewise, nest pairs that were initially March 2000 March January 2000 October 1999

February 2000 nonaggressive typically did not develop aggression. December 1999 November 1999 September 1999 Taken together, these findings suggest that patterns of Figure 5. Temporal pattern of aggression between laboratory nestmate discrimination can remain stable over time, nests and the field nests from which they were collected. Closed even when environmental factors are tightly controlled. circles include nests from all eight locations for which nests were A loss of intraspecific aggression under laboratory condi- maintained in the laboratory. Open circles exclude data from tions is often observed in social insects and is commonly Sweetwater 3A (see Results). Error bars represent one standard error. attributed to reduced variation in environmentally derived odours or the necessity of direct contact between nests (Stuart 1987; Breed 1998; Boulay et al. 2000; Chen & Nonacs 2000). However, many studies, including this 4 one, have demonstrated that aggression among nests is maintained or can even increase when controlling for environmental differences in the laboratory (Jutsum et al. 1979; Le Moli et al. 1992; Heinze et al. 1996; Holway et al. 3 1998; Stuart & Herbers 2000). Aggressive nest pairs showed significant temporal vari- ation in degree of aggression for both field and laboratory 2 comparisons. This variation may result from a number of factors. First, temporal variation in aggression may be related to extrinsic environmental factors such as tem- 1 Y = 8.227 – 7.004 X; R2 = 0.58 poral changes in diet, temperature or moisture. However,

Average aggression between nest pairs Average the role of environmental factors in this study appears 0.6 0.7 0.8 0.9 1.0 relatively minor given that levels of aggression were Correlation of hydrocarbon profiles between nests similar between field and laboratory nest pairs for the first Figure 6. Relationship between the average aggression score 9 months of the study (Fig. 2). Second, temporal variation and the correlation of hydrocarbon profiles between nest pairs. in aggression may also be related to intrinsic factors such Aggression and hydrocarbon data were collected from field nests in as reproductive phenology (Passera & Aron 1993), August 1999. changes in local worker density, or variation in within- nest patterns of relatedness. Although such sources of variation undoubtedly influence nestmate recognition, Hydrocarbon Analysis we found no evidence that levels of aggression corre- sponded with one such measure, the timing of male Across all nest pairs, levels of aggression were nega- release (McCluskey 1963; Markin 1970; A. V. Suarez, tively related to the overall similarity of cuticular hydro- unpublished data). Variation in aggression may also be carbon profiles (Mantel test: z=17.9911, R2=0.5802, related to seasonal cycles of worker production or nest P<0.0001)(Fig. 6). A specific example of this association is activity (Ichinose 1991). For example, levels of aggression illustrated in Fig. 7. Ants from the Mesa and Sweetwater peaked in the autumn when worker densities are highest 3B sites, which never showed aggression towards (Newell & Barber 1913; Markin 1968). Lastly, variation in each other, had similar cuticular hydrocarbon profiles aggression may be influenced by temporal changes in

(Pearson’s correlation: r42=0.994). In contrast, ants from within-nest relatedness resulting from queen turnover the Temecula site consistently fought with ants from and colony contraction and expansion. both Mesa and Sweetwater 3B and had profiles that were An important exception to the prevailing patterns less correlated (Pearson’s correlations: Temecula/Mesa: described above involved the behaviour and hydro- r42=0.711; Temecula/Sweetwater 3B: r42=0.778; Fig. 7). carbon profiles of ants collected at the Sweetwater 3A site. 704 ANIMAL BEHAVIOUR, 64, 5

Mesa

Sweetwater 3B Detector response

Temecula

0 5 10 15 20 25 30 Retention time (min) Figure 7. Sample cuticular hydrocarbon profiles isolated from Argentine ants collected at the Mesa, Sweetwater 3B and Temecula field sites in August 1999. Ants from Mesa and Sweetwater 3B did not show aggression towards one another, while ants from both locations showed aggression towards ants from Temecula. Closed arrows point to the two largest peaks from the Mesa profile. Open arrows point to example hydrocarbon peaks that differed between Mesa/Sweetwater 3B and Temecula.

At the beginning of the study, workers from this location Sweetwater 3A began to fight with ants from these three did not fight against workers from Coast, Mesa and locations and their laboratory counterparts collected Sweetwater 3B. After 6 months, however, ants from from exactly the same site (Figs 2, 3). Although the mechanism responsible for this result remains unclear, several hypotheses can be advanced. First, the field col- ony at Sweetwater 3A may have changed its behaviour. 1.0 For example, a sudden and sustained change in some important environmental factor influencing cuticular 0.8 hydrocarbon profiles (and subsequently nestmate recog- nition) may have influenced the behaviour of these ants. 0.6 A second explanation involves a spatial change in colony boundaries. In this area, two behaviourally antagonistic supercolonies exist in close proximity. It is plausible that 0.4 during the course of this study the territorial boundaries Mesa between these two supercolonies shifted such that the profiles between nests 0.2 Sweetwater 3A point from which we were sampling at Sweetwater 3A Correlation of hydrocarbon contained workers from one supercolony at the start of 0.0 August 1999 December 1999 March 2000 August 2000 the study and workers from the other supercolony after January 2000. If so, we would also expect to see a genetic Figure 8. Correlation coefficients between hydrocarbon profiles change in the colony corresponding to the change in from ants collected in August 1999 and ants collected at three later behaviour. This interesting and unique event warrants time periods for the Mesa and Sweetwater 3A sites. Each point represents the mean (+1 SD) correlation coefficient based on three further scrutiny and is the focus of current investigation. replicates for each time period. The onset of aggression between With the exception of Sweetwater 3A, intraspecific ants from the Sweetwater 3A field site and their laboratory counter- aggression rarely developed between laboratory nests part occurred in January 2000. and the field nests from which they were collected, SUAREZ ET AL.: AGGRESSION AMONG ARGENTINE ANT NESTS 705

Figure 9. Representative hydrocarbon profiles of Argentine ants from Mesa and Sweetwater 3A field sites at four different times during a 1-year period. Open arrows point to the three largest hydrocarbon peaks in ants from Sweetwater 3A during August 2000. These peaks are the same as those identified with open arrows in Fig. 7. Solid arrows point to examples of new hydrocarbon peaks that were only observed in ants from Sweetwater 3A during March and August 2000. until the last 2 months of the study (Fig. 5). prey-derived hydrocarbons can cause aggression in This pattern has several potential explanations. First, Argentine ants (Liang & Silverman 2000). Liang & environmental determinants of nestmate discrimination Silverman (2000) demonstrated that Argentine ant may persist under controlled conditions for up to a year. workers can acquire unique hydrocarbons from some However, given the relatively rapid loss of intraspecific prey and that aggression between (and within) nests aggression in some laboratory studies (e.g. Stuart 1987; develops when one nest is fed brown-banded cock- Chen & Nonacs 2000), this explanation seems unlikely. roaches. However, nest isolation via prey-derived hydro- Second, the development of aggression between labora- carbons may be rare in the field because the diets of tory nests and their field counterparts at the end of the individual Argentine ant nests are probably diverse. experiment may have resulted from changes in colony Moreover, unlike the pronounced response produced by structure associated with the subset of queens that were brown-banded cockroaches, aggressive behaviour among brought into the laboratory. Because Argentine ant nests does not develop when other prey items are used workers typically live less than 1 year (Newell & Barber (Liang et al. 2001). Cuticular hydrocarbons may also vary 1913; Giraud et al. 2002), our laboratory colonies had seasonally (Vander Meer et al. 1989; Nielsen et al. 1999), near complete worker turnover by the end of the exper- suggesting that either factors related to the environment iment. Therefore, long-lasting environmental effects will or reproduction can influence hydrocarbon profiles. Our diminish as field-collected workers are replaced with results from the Mesa site (Fig. 9) suggest that hydro- laboratory-reared workers. Moreover, patterns of within- carbon profiles show little temporal variation throughout nest relatedness will change, as field-collected workers the course of a year. However, it would be of great interest are replaced with workers that eclose within the labora- to determine in more detail whether temporal variation tory, reflecting the genetic makeup of the laboratory in aggression is mirrored by temporal variation in queens. hydrocarbon profiles. As in other ant species, an association exists between The behavioural patterns seen in the laboratory cuticular hydrocarbon profiles and aggression in the portion of our study suggest that the environmental Argentine ant. Specifically, nest pairs with highly similar component of nestmate discrimination may be of minor hydrocarbon profiles did not show aggression, whereas importance. However, several limitations prevent us nest pairs with divergent profiles tended to fight (Fig. 6). from fully exploring the mechanisms of nestmate dis- The underlying causes of cuticular hydrocarbon variation criminatory ability in this system. First, increased queen are poorly understood, although both environmental number may compromise the ability of nestmates to and genetic factors probably play a role. For example, recognize foreign individuals (Ho¨lldobler & Wilson 706 ANIMAL BEHAVIOUR, 64, 5

1977) as has been shown in some ants (Keller & Passera Bonavita-Cougourdan, A., Clement, J. L. & Lange, C. 1987. 1989; Provost 1989; Starks et al. 1998a; Morel et al. Nestmate recognition: the role of cuticular hydrocarbons in the 1990; but see Crosland 1990; Stuart 1991). Argentine ant Camponotus vagus. Scop. Journal of Entomological Science, 22, ant nests can have hundreds of queens, in both native 1–10. Bond, W. & Slingsby, P. 1984. Collapse of an ant–plant mutualism: and introduced populations (A. V. Suarez, N. D. Tsutsui, the Argentine ant ( humilis) and myrmecochorous D. A. Holway, & T. J. Case, personal observations). Proteaceae. Ecology, 65, 1031–1037. Ideally, queen number should have been constant in Boulay, R., Hefetz, A., Soroker, V. & Lenoir, A. 2000. Camponotus our laboratory study as within-nest relatedness may fellah colony integration: worker individuality necessitates decrease with increasing queen number, particularly if frequent hydrocarbon exchanges. Animal Behaviour, 59, queens are multiply mated (but see Krieger & Keller 1127–1133. 2000). Moreover, aggression between nests may be influ- Bourke, A. F. G. & Franks, N. R. 1995. Social Evolution in Ants. enced by intranest relatedness as well as the genetic Princeton, New Jersey: Princeton University Press. similarity among nests (Pirk et al. 2001; Tsutsui & Case Breed, M. D. 1998. Recognition phermones of the honey . 2001). Our study also did not examine context- BioScience, 48, 463–470. Breed, M. D. & Bennett, B. 1987. Kin recognition in highly eusocial dependent recognition (i.e. proximity to one’s colony) insects. In: Kin Recognition in (Ed. by D. J. C. Fletcher & (Starks et al. 1998b). Our behavioural assay was per- C. D. Michener), pp. 243–285. New York: J Wiley. formed in a neutral environment in contrast to other Cammell, M. E., Way, M. J. & Paiva, M. R. 1996. Diversity and studies that place one worker (or a set of workers) in the structure of ant communities associated with oak, pine, context of defenders and the other(s) as invaders (e.g. and arable habits in Portugal. Insectes Sociaux, 43, 37–46. Chen & Nonacs 2000; Stuart & Herbers 2000; Pirk et al. Carlin, N. F. 1989. Discrimination between and within colonies of 2001). Directional or asymmetric aggression may be social insects: two null hypotheses. Netherlands Journal of Zoology, prevalent and important in untangling mechanisms 39, 86–100. behind nestmate discrimination (Stuart & Herbers 2000; Carlin, N. F. & Ho¨lldobler, B. 1986. The kin recognition system of Pirk et al. 2001). carpenter ants (Camponotus spp.); I. Hierarchical cues in small In this study we have attempted to increase our colonies. Behavioral Ecology and Sociobiology, 19, 123–134. Chen, J. S. C. & Nonacs, P. 2000. Nestmate recognition and understanding of the underlying causes of intraspecific intraspecific aggression based on environmental cues in Argentine aggression in a species in which aggression is typically ants (: Formicidae). Annals of the Entomological absent from introduced populations. Given that intro- Society of America, 93, 1333–1337. duced populations of invasive ants often lack intra- Crosland, M. W. J. 1990. The influence of queen, colony size, and specific aggression (Morel et al. 1990; Passera 1994; worker ovarian development on nest mate recognition in the ant Holway & Suarez 1999) it will be of great interest to Rhytidoponera confusa. Animal Behaviour, 39, 413–425. learn more about the nestmate recognition systems of Gamboa, G. J., Reeve, H. K. & Pfennig, D. W. 1986. The evolution these species (e.g. Obin & VanderMeer 1988). Such and ontogeny of nestmate recognition in social wasps. Annual information may be useful in the development of con- Review of Entomology, 31, 431–454. trol strategies that try to induce intraspecific aggression Giraud, T., Pederson, J. S. & Keller, L. 2002. Evolution of super- colonies: the Argentine ants of southern Europe. Proceedings of the in invasive ants, either through genetic (Suarez et al. National Academy of Sciences, U.S.A., 99, 6075–6079. 1999; Tsutsui et al. 2000) or chemical (Silverman & Hamilton, W. D. 1964. The genetical evolution of social behavior. I Liang 2001) means. and II. Journal of Theoretical Biology, 7, 1–52. Haverty, M. I., Grace, J. K., Nelson, L. J. & Yamamoto, R. T. 1996. Intercaste, intercolony, and temporal variation in cuticular Acknowledgments hydrocarbons of Coptotermes formosanus Shiraki (Isoptera: Phinotermitidae). Journal of Chemical Ecology, 22, 1813–1834. We would like to thank J. Baez and K. Royer for help with Heinze, J., Foitzik, S., Hippert, A. & Holldobler, B. 1996. the aggression assays. 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